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LD marker/structure-like proteins have also been extended using proteomics from
mammalian and
Drosophila
perilipin family (
Kimmel, Brasaemle, McAndrews-
Hill, Sztalryd, & Londos, 2010
)to
C. elegans
DHS-3 (
Zhang et al., 2012
), green al-
gae MLDP (
Moellering & Benning, 2010
), and bacterial MLDS (
Ding et al., 2012
).
However, more research needs to be conducted to further elucidate the full array of
properties and functions of this organelle. The presented method allows for a stan-
dardized isolation and study of LDs.
Despite the dramatic progress of LD protein identification, some uncertainties
remain. A major issue is the purity of isolated LDs, like other organelles and cell
fractions, constant modification of the isolation methods such as varying centrifuga-
tion speed and duration, washing length and times, buffer properties and components
including buffer type, buffer pH, ion type and strength, as well as detergent types and
overall concentration. Although these modifications significantly improve the purity
of isolated LDs, ER proteins and mitochondrial proteins are often detected (i.e.,
mouse skeletal muscle LD isolation) by proteomic analysis. On the other hand,
the presence of ER contaminant is debatable. LDs have been previously proposed
to be constantly associated with ER in yeast (
Goodman, 2008
) while the existence
of
the ER-associated LDs and cytosolic LDs has been speculated (
Hayashi & Su,
2003
). In addition, an organelle with both partial LD and partial ER elements was
hypothesized (
Fujimoto, Ohsaki, Cheng, Suzuki, & Shinohara, 2008
). Nevertheless,
physiological contact between LDs and ER results in the presence of ER proteins in
LD proteomes and has become an accepted fact of LD isolation. In fact, the physical
contact between LDs and other organelles has been observed by electron micro-
scope, suggesting that similar to ER, finding proteins of other organelles in isolated
LDs may be due to their physiological interaction, rather than the isolation technique
efficacy. If so, the coisolation of LDs with other organelles may be a useful quan-
tifiable measure of their interaction, when isolated under highly stringent conditions.
Nevertheless, LDs free of other organelle proteins do still occur and are essential for
the identification and characterization of various LD proteins. A new method termed
protein correlation profiles (PCP) was reported recently. PCP aims to increase fidel-
ity of LD proteome (
Krahmer et al., 2013
), as the method analyzes more than four
cellular fractions that we have established previously (see
Fig. 1.1
)(
Bartz, Zehmer,
et al., 2007; Ding et al., 2012; Zhang et al., 2012
). Moreover, LD proteomes and more
than simply mixtures of LDs as LDs are very diverse in size and protein composition,
with the exception of the unilocular LD found in adipocytes. Interestingly, some pro-
teins may not be localized in all LDs, although some are localized. Thus, to obtain
more accurate LD proteomes, current isolated LDs need to be further purified using
more complicated means such as immune-isolation and sorting based on size or sur-
face marker. In summary, the current LD isolation method needs to be improved to
effectively separate LDs from other organelles more efficiently and further sort sub-
populations of LDs by size and by protein composition.
Only adipocytes contain a unilocular LD per cell, and other types of cells have mul-
tiple LDs with varying size distribution. Moreover, another difficulty in obtaining
proper LD proteome may arise from the isolation procedure. During the isolation pro-
cess, LDs are separated from other cellular fractions using centrifugation. LDs have a
